Amyloid specific binding peptides and detecting abeta peptide

ABSTRACT

Phage peptide display technology was used to identify peptides that bind specifically to the amyloid form of the Aβ 1-40  peptide. Peptides with similar structural features and bind to the amyloid form of Aβ 1-40  but not to monomeric Aβ 1-40 , are provided. Such peptides are useful as carrier molecules to deliver therapeutic and diagnostic reagents to amyloid plaques.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is filed under 35 U.S.C. §371 and claims priority toInternational Application Serial No. PCT/US2004/010939, filed Apr. 7,2004, which claims priority under 35 U.S.C. §119 from provisionalapplication Ser. No. 60/461,168, filed Apr. 7, 2003, the disclosure ofwhich is incorporated herein by reference.

STATEMENTS REGARDING FEDERALLY SPONSORED RESEARCH

The invention was funded in part by Grant No. NS37214 awarded by theNational Institutes of Health.

TECHNICAL FIELD

This invention relates generally to peptides that interact with proteinaggregates associated with a disease state, and more specifically topeptides that interact with amyloid plaques containing amyloid β (Aβ)peptide.

BACKGROUND

Alzheimer's disease (AD) is the major cause of dementia in the elderly,affecting approximately 3-4 million people in the United States alone.The decline of cognitive abilities in AD is associated with pathologicchanges in the brain, the most prevalent of which are the formation ofamyloid plaques and neurofibrilary tangles (Selkoe, Physiol. Rev.81:741-766, 2001). Amyloid plaques in AD brains form at far greaternumbers than in normal individuals. While amyloid plaques contain manyproteins, they have as their principal constituent the 4 kDa amyloid-β(Aβ) peptide (Kang et al., Nature 325:733-736. 1987). The formation ofthe Aβ peptide, and thereby Aβ amyloid, arises from aberrant processingof the amyloid precursor protein (APP). A number of studies support theidea that Aβ is itself neurotoxic, and therefore the high concentrationof Aβ peptide in amyloid plaques may seed the generalized degenerationof neurons in surrounding areas (Morris and Price, J. Mol. Neurosci.17:101-118, 2001).

One approach to inhibiting AD would appear to be to inhibit theproteases, in particular the K- and β-secretase, that produce the Aβpeptide. However, individuals without AD also have plaques, and as someAβ peptide is produced in people without AD. Therefore, such peptideprocessing may be a byproduct of necessary protease functions andinhibiting APP processing may have unwanted and toxic consequences.

Another approach would be to design therapies that would eithereliminate the toxic aspects of amyloid plaques or remove plaques fromthe brain altogether. For example, Aβ toxicity is associated with thegeneration of reactive oxygen species (Parks et al., J. Neurochem.76:1050-1060, 2001) and with the accumulation of heavy metals (Cherny etal., Neuron 30:665-676, 2001). Therefore, the creation of a reducing orchelating environment locally at amyloid plaques may inhibit thetoxicity associated with Aβ in these areas. Because many proteins inaddition to Aβ accumulate in amyloid plaques, the activation ofproteases may also aid in plaque removal or lessen plaque number orplaque size. Blocking of the cellular receptors that mediate Aβ toxicityin neurons may also have a therapeutic benefit.

These approaches would be greatly facilitated by the ability to targettherapeutics directly to amyloid plaques. One way to do this would be todevelop reagents that specifically bind Aβ amyloid and can be conjugatedwith therapeutic or diagnostic molecules. It is an object herein, amongother objects, to provide reagents that specifically react with Aβamyloid, diagnostic assays using such reagents, and methods forpreparing reagents for identifying disease causing forms of otheramyloid proteins and other disease-associated conformation dependentproteins.

SUMMARY

Provided herein are peptides that specifically react with a targetpolypeptide, which is the aberrant form of a polypeptide associated witha disease of protein aggregation (a disease involving a conformationallyaltered protein), such as amyloid diseases.

In one embodiment, an isolated polypeptide comprising the amino acidsequence Y (Trp/Phe) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ (Trp/Phe) Xaa₆ Xaa₇(Trp/Phe) Z (SEQ ID NOs:17-20) is provided. Y, which may or may not bepresent, is a peptidic structure containing at least one cysteineresidue and having the formula (Xaa)_(n). Xaa is any amino acid residueand n is an integer from 1 to 20. A which may or may not be present, isa peptidic structure containing at least one cysteine residue and havingthe formula (Xaa)_(n), wherein Xaa is any amino acid residue and n is aninteger front 1 to 20. The amino acid residues of in Xaa₁ through Xaa₇can be any amino acid and the amino acid residues of Xaa₁ through Xaa₃are positively charged.

In another embodiment, an isolated polypeptide comprising the amino acidsequence Y (Trp/Phe) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ (Trp/Phe) Xaa₆ Xaa₇ Xaa₈(Trp/Phe) Z (SEQ ID NOs:21-24) is provided. Y, which may or may not bepresent, is a peptidic structure containing at least one cysteineresidue and having the formula (Xaa)_(n). Xaa is any amino acid residueand n is an integer from 1 to 20. Z, which may or may not be present, isa peptidic structure containing at least one cysteine residue and havingthe formula (Xaa)_(n), wherein Xaa is any amino acid residue and n is aninteger from 1 to 20. The amino acid residues of Xaa₁ through Xaa₈ isany amino acid, and at least two of the residues of Xaa₁ through Xaa₅are positively charged.

In other embodiments, a is 1-15, 1-10, 1-5, or 1-3 residues in length.In addition, the cysteine in the Y peptidic structure and the cysteinein the Z peptidic structure are intramolecularly cross linked via adisulfide bond.

Isolated polypeptides comprising the amino acid sequence set forth inSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, areprovided. Isolated polypeptides consisting of the sequence set forth inSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, arealso provided.

Nucleic acid sequences encoding a polypeptide of the invention are alsoprovided. Vectors containing such nucleic acids, and cells containingsuch vectors, are also provided.

In addition, hybrid molecules, such as hybrid polypeptides, with suchspecificity are provided. The hybrid polypeptides include a peptidemotif that specifically interacts with the target polypeptide (e.g., theamyloid form of the Aβ peptide) and that is inserted into a scaffold,such as a portion of an antibody or an enzyme or other suitablerecipient, such that the resulting hybrid molecule specifically binds toconformation of the protein and not to another conformation of theprotein (e.g., the amyloid form of the Aβ peptide and not the monomericform of the Aβ peptide). Typically, the targeted conformation is theconformation involved in a disease. The polypeptide motif is insertedinto the scaffold such that any desired function of the scaffold isretained and the inserted motif as presented retains it ability tospecifically bind to the target. The selected scaffold can be exploitedfor its activities or binding sites to aid or permit detection ofcomplexes between the motif and the target polypeptide. The scaffold caninclude, for example, neuroprotective agents to make amyloid plaquesless toxic, amyloid destroying molecules to eliminate plaques, reagentsthat prevent amyloid plaque formation, or reagents useful forspecifically imaging amyloid plaques in brain tissue.

Methods for producing peptides for detection or diagnosis ofconformationally altered protein diseases and for treatment thereof areprovided. Such diseases include, but are not limited to, Alzheimer'sDisease (AD); Creutzfeldt-Jakob disease, including variant, sporadic andiatrogenic, scrapie and bovine spongiform encephalopathy; Type IIDiabetes (islet amyloid peptide); Huntington's Disease; immunoglobulinamyloidosis; reactive amyloidosis associated with chronic inflammatorydisease, e.g., inflammatory arthritis, granulomatous bowel disease,tuberculosis and leprosy; hereditary systemic amyloidosis associatedwith autosomal dominant inheritance of variant transthyretin (a.k.a.,prealbumin) gene; ALS; Pick's Disease; Parkinson's disease;Frontotemporal dementia; Diabetes Type II; Multiple myeloma; Plasma celldyscrasias; Familial amyloidotic polynueuropathy; Medullary carcinoma ofthyroid; chronic renal failure; congestive heart failure; senile cardiacand systemic amyloidosis; chronic inflammation; atherosclerosis;familial amyloidosis and other such diseases.

The hybrid polypeptides can be used as reagents to detect the presenceof the target polypeptide in a sample, such as a body fluid, tissue ororgan or a preparation derived therefrom, and in drug screening assaysto identify compounds that antagonize or agonize (i.e., modulate) theactivity of a target polypeptide or that competitively inhibitinteraction thereof with an infectious or disease-causing form of atarget polypeptide, such as the amyloid form of Aβ peptide. The hybridmolecules also can be used as therapeutics. Since they specifically bindto a target polypeptide, they can be used to inhibit its activity, suchas preventing or reducing the activity that results in proteinaggregation or the conformation change leading to a deleterious effect.For example, as a therapeutic for treatment of diseases of proteinaggregation a hybrid polypeptide can interrupt the polymerization oraggregation characteristic of disease pathogenesis.

In an exemplary embodiment, hybrid polypeptides that specifically reactwith the amyloid form of the amyloid β (Aβ) peptide are provided. Inaddition, hybrid polypeptides that bind specifically todisease-associated conformations of the Aβ peptide are provided.

In an exemplary embodiment, methods for detection of the amyloid form ofthe amyloid β (Aβ) peptide in a sample, such as a body fluid, tissue ororgan from an animal, are provided. The methods are effected in solutionphase or by providing the reagents or sample bound directly orindirectly to a solid support. Complexes between the reagents providedherein and the target polypeptides in the sample are detected.

Also provided are anti-idiotype antibodies (monoclonal or polyclonal)that are produced by immunizing a suitable animal with a polypeptide orantibody or fragment thereof that recognizes a peptide disclosed herein,monoclonal antibody Fab fragments or other inhibitory antibodies.Anti-idiotype antibodies raised against the combining sites ofinhibitory antibodies or Fabs, can generate antibodies that recognizenative the amyloid form of Aβ peptide. Such anti-idiotype antibodies canbe used in all of the diagnostic, prognostic, therapeutic and screeningmethods that the hybrid polypeptides also provided herein are used.Methods for preparing such anti-idiotype antibodies by immunizing with apolypeptide or antibody or fragment thereof that recognizes the amyloidform of Aβ peptide from about amino acid 1 to amino acid 40, also areprovided.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts staining of amyloid Aβ₁₋₄₀ by phage peptides.

FIG. 2 depicts immunoblotting of monomeric Aβ₁₋₄₀ by phage peptides.

FIG. 3 depicts recombinant Aβ-binding peptide binding with high affinityto Aβ₁₋₄₀ amyloid in vitro.

FIG. 4 depicts specific binding of Thio-Aβ to amyloid plaques inAlzheimer's disease brain.

FIG. 5 depicts binding of synthetic peptides to Aβ₁₋₄₀ amyloid in vitro.

FIG. 6 depicts specific staining amyloid plaques in Alzheimer's diseasebrain with a synthetic peptide.

FIG. 7 depicts a model of uses for the Aβ₁₋₄₀ binding-peptides.

FIG. 8 depicts entry of amyloid binding peptide into the brain afterintravenous injection.

DESCRIPTION

Peptides, nucleic acids encoding the peptides, and methods of using thepeptides or nucleic acids to diagnose and/or treat diseases associatedwith plaque formation in brain tissue, such as Alzheimer's Disease (AD),are provided. For example, the peptides of the invention canspecifically target the amyloid form of the Aβ₁₋₄₀ peptide in plaques ofAlzheimer's patients.

Peptides

Provided herein are peptides that specifically bind to amyloid form ofthe Aβ peptide and methods of preparing such polypeptides and hybridpolypeptides that comprise a peptide of the invention and additionalamino acids. The peptides of the invention bind to disease-causingconformers of conformationally altered protein diseases (diseasesinvolving protein aggregation). As used herein, conformationally alteredprotein disease (or a disease of protein aggregation or a disease ofprotein conformation) refers to diseases associated with a protein orpolypeptide that has a disease-associated conformation. Abnormal proteinconformation, including, for example, misfolding and aggregation, canlead to a loss or alteration of biological activity. Abnormal proteinconformation, including misfolding and aggregation is a causative agent(or contributory agent) in a number of mammalian, including, but are notlimited to, Alzheimer's disease, prion spongiform encephaplopathies,such as bovine spongiform encephalopathy, scrapie of sheep, Kuru andCreutzfeldt-Jakob disease of humans, including variant, sporadic andiatrogenic, and amyotrophic lateral sclerosis (ALS).

In one embodiment, an isolated polypeptide comprising the amino acidsequence Y (Trp/Phe) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ (Trp/Phe) Xaa₆ Xaa₇(Trp/Phe) Z (SEQ ID NOs: 17-20) is provided. Y, which may or may not bepresent, is a peptidic structure containing at least one cysteineresidue and having the formula (Xaa)_(n). Xaa is any amino acid residueand n is an integer from 1 to 20. Z, which may or may not be present, isa peptidic structure containing at least one cysteine residue and havingthe formula (Xaa)_(n), wherein Xaa is any amino acid residue and n is aninteger from 1 to 20. The amino acid residues of in Xaa₁ through Xaa₇can be any amino acid and the amino acid residues of Xaa₁ through Xaa₅are positively charged.

In another embodiment, an isolated polypeptide comprising the amino acidsequence Y (Trp/Phe) Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ (Trp/Phe) Xaa₆ Xaa₇Xaa₈(Trp/Phe) Z (SEQ ID NOs: 21-24) is provided. Y, which may or may notbe present, is a peptidic structure containing at least one cysteineresidue and having the formula (Xaa)_(n). Xaa is any amino acid residueand n is an integer from 1 to 20. Z, which may or may not be present, isa peptidic structure containing at least one cysteine residue and havingthe formula (Xaa)_(n), wherein Xaa is any amino acid residue and n is aninteger from 1 to 20. The amino acid residues of Xaa₁ through Xaa.sub.8is any amino acid, and at least two of the amino acid residues of Xaa₁through Xaa₅ are positively charged.

In other embodiments, a is 1-15, 1-10, 1-5 or 1-3 residues in length. Inaddition, the cysteine in the Y peptidic structure and the cysteine inthe Z peptidic structure are intramolecularly cross linked via adisulfide bond.

A “peptidic structure”, as used herein, is an optional portion of apeptide or polypeptide comprising modified or unmodified amino acids. Apeptidic structure contains at least one cysteine residue that can forma disulfide bond with another cystein residue. The cysteine residuescontained in the Y or Z peptidic structures can be positioned anywherewithin the structure as long as they can form a disulfide bond.

Isolated polypeptides comprising the amino acid sequence set forth inSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, areprovided. Isolated polypeptides consisting of the sequence set forth inSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, arealso provided.

Peptides and polypeptides of the invention include those containingconservative amino acid substitutions. Such peptides and polypeptidesare encompassed by the invention provided the peptide or polypeptide canbind to the amyloid form of Aβ peptide. As used herein, suitableconservative substitutions of amino acids are known to those of skill inthis art and can be made generally without altering the biologicalactivity of the resulting molecule. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity(see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition,1987, The Benjamin/Cummings Pub. co., p. 224). Such substitutions can bemade in accordance with those set forth in TABLE 2 as follows:

Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln (SEQ ID NO: 25); His Cys (C)Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala (SEQ ID NO: 26); Pro His (H)Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (N)Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) Ser Trp (W) TyrTyr (Y) Trp (SEQ ID NO: 27); Phe Val (V) Ile; Leu.

Other substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

Provided are peptides and polypeptides that preferentially(specifically) bind to one conformer (generally the disease-associatedconformer) with greater affinity, typically at least 0.5, 1, 2, 3, 5,10-fold or greater, than to the other conformer. Also contemplated arepeptides-containing deletions of one or more amino acids that result inthe modification of the structure of the resultant molecule but do notsignificantly altering its ability to bind to one conformer, such asamyloid form of the Aβ peptide, to form a plaque-protein complex.

Nucleic Acid Molecules

Nucleic acid molecules encoding any of the peptides, polypeptides orhybrid polypeptides provided herein are provided. Such molecules can beintroduced into plasmids and vectors for expression in suitable hostcells.

As used herein, the term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides such as deoxyribonucleic acid (DNA), andribonucleic acid (RNA) as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives or combinations thereof. The termshould be understood to include, as equivalents, derivatives, variantsand analogs of either RNA or DNA made from nucleotide analogs, single(sense or antisense) and double-stranded polynucleotides.Deoxyribonucleotides include deoxyadenosine, deoxycytidine,deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.

Plasmids and vectors containing the nucleic acid molecules also areprovided. Cells containing the vectors, including cells that express theencoded proteins are provided. The cell can be a bacterial cell, a yeastcell, a fungal cell, a plant cell, an insect cell or an animal cell.Methods for producing a hybrid polypeptide, for example, growing thecell under conditions whereby the encoded polypeptide is expressed bythe cell, and recovering the expressed protein, are provided herein. Thecells are used for expression of the protein, which can be secreted orexpressed in the cytoplasm. The hybrid polypeptides also can bechemically synthesized using standard methods of protein synthesis.

Hybrid Molecules

For disease of protein conformation the same protein (or a portionthereof) exhibits more than one isoform (conformer) such that at leastone form is causative of a disease, such as the amyloid form of the Aβpeptide, or is involved in the disease. For purposes of diagnosis,prognosis, therapy and or drug screening it is advantageous to havemolecules that specifically interact (i.e. react with greater affinity,typically at least, 2-, 5-10-fold, generally at least about 100-fold)with a disease-associated conformer than with a benign (non-diseaseinvolved) conformer (or vice versa). Hence provided herein are peptidesthat specifically react with one conformer of a protein (i.e., theamyloid form of the Aβ peptide). Typically the molecules interact with adisease-associated conformer. A hybrid molecule of the inventionincludes a peptide or polypeptide that binds to the amyloid form of Abpeptide, and a scaffold molecule. The scaffold molecule can include adiagnostic or therapeutic reagent. The therapeutic or diagnostic reagentcan be a polypeptide, small molecule or compound.

In particular, provided herein are hybrid molecules, such as hybridpolypeptides, that include a peptide or polypeptide provided herein, andadditional amino acid residues (typically, 5, 10, 15, 20, 30, 40, 50,100 or more) such that the resulting hybrid molecule specificallyinteracts with the amyloid form of the Aβ peptide). The motif can bemodified, such as by replacing certain amino acids or by directed andrandom evolution methods, to produce motifs with greater affinity.

As used herein, a hybrid polypeptide refers to a polypeptide thatincludes regions from at least two sources, such as from an antibody orenzyme or other scaffold that can be a recipient, and a binding motif,such as a polypeptide or peptide that binds to an amyloid form of the Aβpeptide.

Thus, among the hybrid molecules provided herein are hybrid molecules,particularly hybrid polypeptides, that are produced by grafting abinding motif (e.g., peptide) from one molecule into a scaffold, such asan antibody or fragment thereof or an enzyme or other reporter molecule.The hybrid polypeptides provided herein, even the hybridimmunoglobulins, are not antibodies per se, but are polypeptides thatare hybrid molecules containing a selected motif (e.g., a peptide thatbinds to the amyloid form of the Aβ peptide) inserted into anotherpolypeptide such that the motif retains or obtains the ability to bindto a protein involved in disease of protein aggregation. The hybridpolypeptides can include portions of antibodies or other scaffolds, butthey also include a non-immunoglobulin or non-scaffold portion graftedtherein. The non-immunoglobulin portion is identified by its ability tospecifically bind to a targeted polypeptide isoform. The hybridpolypeptide can specifically bind to the targeted infectious ordisease-related or a selected isoform of a polypeptide as monomer withsufficient affinity to detect the resulting complex or to precipitatethe targeted polypeptide.

The scaffold is selected so that insertion of the motif therein does notsubstantially alter (i.e., retains) the desired binding specificity ofthe motif. The scaffold additionally can be selected for its properties,such as its ability to act as a reporter.

Methods for production of hybrid molecules that specifically interactwith a one form of a conformer of a protein associated with a disease ofprotein conformation or involving protein aggregation are provided. Inthese methods a polypeptide motif from the protein is inserted into ascaffold such that the resulting molecule exhibits specific binding toone conformer compared to other conformers. In particular, the hybridmolecule can exhibit specific binding to the amyloid form of the Aβpeptide.

Peptides of the invention have been shown to bind to AD plaques in vitroand in vivo. The peptides can be incorporated in to a scaffold thatcomprises additional amino acid sequences and/or compounds. The hybridmolecule can then be used to label or treat the plaques associated withAβ amyloid. The polypeptides, nucleic acids encoding the polypeptides,and methods of using the polypeptides or nucleic acids can be used toidentify, diagnose and/or treat disorders associated with plaqueformation in brain tissue.

Any molecule, such as a polypeptide, into which the selected polypeptidemotif is inserted (or linked) such that the resulting hybrid polypeptidehas the desired binding specificity, is contemplated for use as part ofthe hybrid molecules herein. The polypeptides can be inserted into anysequence of amino acids that at least contains a sufficient number (10,20, 30, 50, 100 or more amino acids) to properly present the motif forbinding to the targeted amyloid plaque. The purpose of the scaffold isto present the motif to the targeted polypeptide in a form that bindsthereto. The scaffold can be designed or chosen to have additionalproperties, such as the ability to serve as a detectable marker or labelor to have additional binding specificity to permit or aid in its use inassays to detect particular isoforms of a target protein (e.g., theamyloid form of the Aβ peptide) or for screening for therapeutics orother assays and methods.

The scaffolds include reporter molecules, such as fluorescent proteinsand enzymes or fragments thereof, and binding molecules, such asantibodies or fragments thereof. Selected scaffolds include all orportions of antibodies, enzymes, such as luciferases, alkalinephosphatases, β-galactosidase and other signal-generating enzymes,chemiluminescence generators, such as horseradish peroxidase;fluorescent proteins, such as red, green and blue fluorescent proteins,which are well known; and chromogenic proteins.

The peptide motif is inserted into the scaffold in a region that doesnot disturb any desired activity. The scaffolds can include otherfunctional domains, such as an additional binding site, such as onespecific for a second moiety for detection.

Diagnostic and Therapeutic Methods

Methods for detecting an isoform of polypeptide associated with adisease of protein aggregation are provided. The methods include thesteps of contacting a sample suspected of containing the isoform with ahybrid polypeptide that specifically binds to the isoform, such as theamyloid form of Aβ peptide, and detecting binding of the polypeptide.Detection can be effected by any method known to those of skill in theart, including radiolabel, color or fluorescence detection, massspectrometry and other detection methods. For example, the hybridpolypeptide can be detectable labeled or can contain a fluorescent orchromogenic moiety or moieties or can be a fluorescent or chromogenicpeptide or other reporter, such as an enzyme, including a luciferase(from Renilla, Aequora and from other deep sea creatures, from bacteriaor insects) or other enzymatic label. Alternatively, a label, such as afluorescent protein or enzyme can serve as a scaffold into which themotif is inserted, such that the enzymatic activity or fluorescence isretained. Also, the hybrid polypeptide can include additional bindingsites to capture antibodies or nucleic acids or other detectablemoieties.

In one embodiment, a method for identifying the disease-causing form ofa target polypeptide in cells is provided. The hybrid polypeptidespecific for the target is detectably labeled, such as fluorescentlylabeled or inserted into a fluorescent protein or a luciferase, andcontacted with a sample, such as a blood sample. Labeled cells areidentified, such as by flow cytometry and scanning cytometry. Methodsand instruments for identifying very low concentrations of labeled cellsamong unlabeled cells are available (see, e.g., Bajaj et al. (2000)Cytometry 39:285-294, published U.S. application Ser. No. 09/123,564,published as US2002018674, and instrumentation commercialized by Q3DM,LLC, San Diego). In an alternative embodiment, label the hybridpolypeptides that interact with distinct epitopes, such as hybridpolypeptides containing residues from 136-158 and 89-112, with differentcolor dyes. The resulting labeled hybrid polypeptides, such as twopolypeptides, are mixed with cells to be tested simultaneously orsequentially. Association of both colors with a single cell, provides aself-confirmatory assay. For example, peptides that bind to the amyloidform of Aβ peptide (or portions thereof sufficient to interact with anepitope, such as at least amino acids 1-40 of the Aβ peptide, aregrafted into for into different florescent protein, such as a greenfluorescent proteins with distinct emission spectra will achieve thesame double labelling of single cells.

Diseases diagnosed or detected include amyloid diseases such as,Alzheimer's Disease, Type II Diabetes, Huntington's Disease,immunoglobulin amyloidosis, reactive amyloidosis associated with chronicinflammatory disease, e.g., inflammatory arthritis, granulomatous boweldisease, tuberculosis and leprosy, hereditary systemic amyloidosisassociated with autosomal dominant inheritance of variant transthyretingene, ALS, Pick's Disease, Parkinson's disease, Frontotemporal dementia,Diabetes Type II, Multiple myeloma, Plasma cell dyscrasias, Familialamyloidotic polynueuropathy, Medullary carcinoma of thyroid; chronicrenal failure, congestive heart failure, senile cardiac and systemicamyloidosis, chronic inflammation, atherosclerosis and familialamyloidosis.

EXAMPLES

A phage peptide library encoding 5×10⁷ random 20 amino acid sequenceswas used to pan for binding to Aβ₁₋₄₀ amyloid and against adherence totissue culture plastic. We decided to use a library that would becysteine cross-linked at its base so as to increase the chance that theinserted peptide would have some tertiary structure. In addition, eventhough phage libraries with larger peptide inserts contain fewer of thetotal number of possible peptide combinations, we surmised that longersequences would be required to identify high affinity binding motifs forthe 40 amino acid Aβ peptide. After three rounds of positive panningagainst the amyloid form of the Aβ₁₋₄₀ peptide, and two rounds ofnegative panning against tissue culture plastic, ten individual cloneswere sequenced and compared these to ten randomly picked sequences fromthe starting library (Table 1). Multiple clones of only two phagesequences that adhered to the Aβ₁₋₄₀ amyloid were identified.

The identified sequences had 3-fold more bulky hydrophobic residues(phenylalanine or tryptophan) than did sequences randomly picked fromthe starting library. Selected sequences shared bulky hydrophobic aminoacids that were spaced at even intervals [(W/F)X₅(W/F)X_(2/3)(W/F)] (SEQID NOs:1 and 33, respectively), and had two positively charged residues(and no negatively charged residues) in the X₅ region.

Clones expressing the DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2) sequencestained Aβ₁₋₄₀ amyloid. In contrast, phage clones containing thePGRSPFTGKKLFNQEFSQDQ (SEQ ID NO:3) sequence stained Aβ₁₋₄₀ amyloid lesswell, but the level of staining was still significantly above backgroundlevels. No clones from the starting library stained any Aβ₁₋₄₀aggregates when used at the same concentration, and no signal wasobserved in the absence of phage with secondary antibody alone. Inaddition, no phage clones stained monomeric peptide that had beenimmobilized on nitrocellulose. The Aβ aggregates identified by thesepeptides are larger than individual 2-12 nm Aβ fibrils and more closelyresemble large (1-10 μm) aggregates found in amyloid plaques (Roher etal., Proc. Natl. Acad. Sci. USA 83:2662-2666, 1986) and in somepreparations of Aβ₁₋₄₀ (Stine et al., J. Prot. Chem. 15:193-203, 1996).

No clones expressing peptides from either the starting or the finalpeptide sequences identified monomeric Aβ₁₋₄₀ (FIG. 2). Monoclonalantibodies to Aβ peptide did bind, however, demonstrating propertransfer of the Aβ peptide to nitrocellulose. The lack of staining orblotting of the isolated phage sequences to linear Aβ₁₋₄₀ peptideindicate that they specifically recognize the amyloid form of Aβ₁₋₄₀.

To verify that the DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2) peptide wouldidentify Aβ₁₋₄₀ amyloid independently of its presence in bacteriophage,this peptide was produced in recombinant form as a fusion protein withthioredoxin. Cysteines were engineered at either end of the peptide inthe fusion construct, along with several other residues from the phagecoat protein. The ultimate or penultimate residue was engineered to be aproline to mimic predicted beta turn at either side of the 20 amino acidinsert. This protein was termed Thio-Aβ. Recombinant thioredoxin withoutthe Aβ-peptide binding sequence was termed Thio.

After purification by nickel resin chromatography, binding studies wereperformed on Thio and Thio-Aβ to immobilized Aβ₁₋₄₀ amyloid (FIG. 3).Thio did not bind to Aβ₁₋₄₀ amyloid at any concentration below 200 μM.In contrast, Thio-Aβ bound with high affinity to Aβ₁₋₄₀ amyloid. A Kd of60 nM was measured and binding was saturating at 200 nM. To determine ifThio-Aβ would bind to Aβ amyloid that was present in the amyloid plaquesfound in Alzheimer's disease (AD) brains (FIG. 4), AD and normal brainswere stained with Thio and Thio-Aβ. Thio-Aβ bound specifically toamyloid plaques in AD brains, while it did not bind to normal brainsamples. Staining of neurofibrillary tangles was not evident. Positivestaining of amyloid plaques with Thio-Aβ was seen in AD brains frommultiple subjects and was absent in sections from several normal brainsamples. Concentrations as low as 100 nM yielded positive staining forThio-Aβ. In contrast, Thio did not bind to either normal or AD brain atany of the concentrations used.

Chemically synthesized peptides were tested to identify binding toAβ₁₋₄₀ amyloid (FIG. 5). To test the relative contribution of theflanking (N-terminal and C-terminal) cysteines, peptides weresynthesized that either had or did not have these residues; Bothpeptides were made with an N-terminal biotin label to allowidentification using streptavidin. Biotin-DWGKGGRWRLWPGASGKTEA andBiotin-AECDWGKGGRWRLWPGASGKTEACGP were tested for binding to amyloidAβ₁₋₄₀. Biotin-AECDWGKGGRWRLWPGASGKTEACGP bound Aβ₁₋₄₀ amyloid with a Kdof 320 nM. Biotin-DWGKGGRWRLWPGASGKTEA, which lacks flanking cysteines,showed binding in the 10-80 μM range. These data indicate that theterminal cysteines, which can form a disulfide bond, induce aconformation of the 20 amino acid insert that enhances high affinitybinding to Aβ₁₋₄₀ amyloid.

Staining of AD and non-AD brain was repeated usingBiotin-AECDWGKGGRWRLWPGASGKTEACGP (FIG. 6). In addition, tissue fromorgans (kidney, brain, large bowel, small bowel, and prostate) from anon-AD patient with amyloidosis were stained. As with Thio-Aβ, thesynthetic peptide specifically stained amyloid plaques in AD brain. Nostaining of neurofibrillary tangles was detected. As with binding toAβ₁₋₄₀ amyloid in vitro, slightly higher concentrations were needed forstaining of plaques relative to Thio-Aβ. Concentrations as low as 500 nMgave good staining with the peptide. Simultaneous staining of kidneysections containing non-Aβ amyloid demonstrated that binding wasspecific for Aβ amyloid. Thus, this peptide sequence is a high affinityprobe for Aβ amyloid both in vitro and in vivo, both within and outsidethe context of other recombinant protein sequences.

Chemically synthesized peptides were tested to identify binding toAβ₁₋₄₀ amyloid (FIG. 5). To test the relative contribution of theflanking (N-terminal and C-terminal) cysteines, peptides weresynthesized that either had or did not have these residues; Bothpeptides were made with an N-terminal biotin label to allowidentification using streptavidin. Biotin-DWGKGGRWRLWPGASGKTEA (SEQ IDNO:2) and Biotin-AECDWGKGGRWRLWPGASGKTEACGP (SEQ ID NO:4) were testedfor binding to amyloid Aβ₁₋₄₀. Biotin-AECDWGKGGRWRLWPGASGKTEAC GP (SEQID NO:4) bound Aβ₁₋₄₀ amyloid with a Kd of 320 nM.Biotin-DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2), which lacks flankingcysteines, showed binding in the 10-80 μM range. These data indicatethat the terminal cysteines, which can form a disulfide bond, induce aconformation of the 20 amino acid insert that enhances high affinitybinding to Aβ₁₋₄₀ amyloid.

Staining of AD and non-AD brain was repeated usingBiotin-AECDWGKGGRWRLWPGASGKTEACGP (SEQ ID NO:4) (FIG. 6). In addition,tissue from organs (kidney, brain, large bowel, small bowel, andprostate) from a non-AD patient with amyloidosis were stained. As withThio-Aβ, the synthetic peptide specifically stained amyloid plaques inAD brain. No staining of neurofibrillary tangles was detected. As withbinding to Aβ₁₋₄₀ amyloid in vitro, slightly higher concentrations wereneeded for staining of plaques relative to Thio-Aβ. Concentrations aslow as 500 nM gave good staining with the peptide. Simultaneous stainingof kidney sections containing non-A.beta amyloid demonstrated thatbinding was specific for Aβ amyloid. Thus, this peptide sequence is ahigh affinity probe for A.beta amyloid both in vitro and in vivo, bothwithin and outside the context of other recombinant protein sequences.

Using phage display, at least two cysteine-linked 20 amino acid peptidesequences that bind to the amyloid form of Aβ₁₋₄₀ have been identified.Neither of these sequences bind monomeric Aβ₁₋₄₀, and therefore thesepeptides specifically identify the amyloid form of the Aβ₁₋₄₀ protein.Both of these sequences share a [(W/F)X₅(W/F)X_(2/3)(W/F)] (SEQ ID NOs:1 and 33, respectively) structure in common, and both have twopositively charged (and no negatively charged) amino acids within the X₅region. Therefore, cysteine-linked peptides with this repeatinghydrophobic motif provides a template for the design of other peptidesthat can bind to Aβ amyloid with even higher affinity. Production andpurification of one of these peptide sequences as a fusion protein withthioredoxin (Thio-Aβ), or direct chemical synthesis of the peptide,created a high affinity binding protein for Aβ₁₋₄₀ amyloid in vitro.These reagents also bound specifically to amyloid plaques in Alzheimer'sdisease (AD) brain.

Applications for peptides (FIG. 7) of the invention include synthesizinghybrid molecules comprising such peptides and a scaffold. The scaffoldcan include: 1) molecules designed to inhibit the toxicity of amyloidplaques; 2) anti-oxidants to protect against oxidative damage caused bythe Aβ peptide or to chelators that could inhibit the accumulation oftoxic metals; 3) reagents that degrade plaques such as activators oftissue plasminogen, urokinase-type plasminogen, or matrixmetalloproteases that stimulate the breakdown of amyloid plaque proteins(Tucker et al., J. Neurosci. 20:3937-3946, 2000); 4) reagents thatinhibit plaque formation; 5) radionuclides or other markers to imageamyloid plaques in living patients. Alzheimer's disease is currentlydiagnosed through cognitive measures on patient interview. Thesemeasures are time consuming, and post-mortem analysis of brains iscurrently required for a definitive diagnosis. Thus, there is no way tomeasure to extent of brain pathology in living patients. Such a lack ofa quantitative measure makes it difficult to diagnose the early stagesof the disease and to make determinations as to the efficacy of varioustreatments.

These peptides of the invention can also be used to develop ananti-idiotype vaccine. Since these peptides bind Aβ amyloid, they maymimic the Aβ amyloid binding site of cellular receptors involved inmediating the neurotoxic effects of Aβ. If so, immunization using thesepeptides could stimulate the production of blocking antibodies tocellular binding sites for Aβ amyloid.

Thus, the invention also provides are anti-idiotype antibodies(monoclonal or polyclonal) that are produced by immunizing a suitableanimal with a polypeptide or antibody or fragment thereof thatrecognizes the a peptide of the invention. Anti-idiotype antibodiesraised against the combining sites of inhibitory antibodies or Fabs cangenerate antibodies that recognize native a peptide that binds to theamyloid form of Aβ peptide.

Such anti-idiotype antibodies can be used in all of the diagnostic,prognostic, therapeutic and screening methods that the hybridpolypeptides also provided herein are used. Methods for preparing suchanti-idiotype antibodies are known to those skilled in the art.

The development of small peptides that bind to Aβ may be superior forthe diagnostic and treatment purposes to other molecules that are knownto bind to Aβ peptide. The molecules that can bind to Aβ amyloid can bebroken down into three groups: non-antibody proteins, antibodies, andsmall organic molecules. As the peptide sequences identified herein aresmall and relatively hydrophobic, they can traverse the blood-brainbarrier efficiently. In addition, these peptides are less likely to haveany significant toxicity when compared with small organic molecules andmay in some instances be easier to conjugate with other reagents.

A cysteine-linked phage peptide library encoding 5×10⁷ random 20 aminoacid insertions was obtained. Aβ₁₋₄₀ peptide(DAEFKHDSGTEVHHQKLVFFAEDVGSNKGAIIGLMVGVV) (SEQ ID NO:28) was purchasedfrom Bachem (Torrence, Calif.). Biotinylated anti-sheep M13 phagepolyclonal antibody was purchased from 5 Prime-3 Prime (Boulder, Colo.).Alkaline phosphatase and horseradish peroxidase-coupled streptavidinwere purchased from Boehringer Mannheim (Indianapolis, Ind.) and JacksonImmunochemicals (West Grove, Pa.). Radionucleotides for DNA sequencingwere purchased from Amersham (Piscataway, N.J.). Oligonucleotides werepurchased from Genosys (The Woodlands, Tex.). Recombinant peptides fusedto thioredoxin were made and purified using plasmids and reagents in theHis-Patch ThioFusion expression system from Invitrogen (Carlsbad,Calif.). Anti-Aβ monoclonal antibody (2066) was a generous gift fromEdward Koo (UC San Diego). Synthetic peptides containing an N-terminalbiotin were-synthesized and purified by AnaSpec (San Jose, Calif.).

Phage panning and quantitation: Methods for phage panning andamplification were performed as described by Mazzuchcelli et al. (Blood93:1738, (1999)). Aβ₁₋₄₀ peptide was added at a concentration of 20μg/ml in Tris-buffered saline (TBS pH 7.4) to wells of a 24 well tissueculture plate (Falcon-Becton Dickinson, Franklin Lakes, N.J.). Numerousamyloid fibrils and aggregates were evident on the bottom of the plateafter several days (Stine et al., J. Prot. Chem. 15:193-203 (1996)),however, peptide was left on for five days to allow more completeaggregation of the peptide to its amyloid form. Round 1: Plates werewashed with phage buffer (Hanks balanced salts with 1 mM CaCl₂ and 1 mMMgCl₂, 10 mM Hepes, pH 7.4, and 0.5% BSA) 3 times for 5 minutes each.Both plates were then incubated with 15 μl (5-10 copies of everysequence) of starting library diluted into 300 μl of phage buffer forone hour with rocking on a shaker. Plates were washed 6 times for 5minutes each with phage buffer, and bound phage were eluted with phagebuffer containing 0.5% Tween 20. Eluted phage were incubated withstarved K91 cells for 15-20 minutes at room temperature and grown inLuria Broth (LB) with 1 μg/ml kanamycin for 45 minutes on a bacterialshaker. A portion of this material was used to titer phage using aplaque assay as previously described (Smith and Scott, Methods. Enzymol.217:228-257 (1993)). Infected bacteria were then plated on 15 cm LB-agarplates containing 75 μg/ml kanamycin overnight at 37° C. Bacteria werescraped from plates and collected in 3 ml TBS per plate (pH 7.5).Bacterial suspension was transferred to Nalgene tubes and spun at 9,100g for 10 minutes. Supernatant was collected and phage precipitated in0.15 volumes of PEG/NaCl (16.7% polyethylene glycol-8000/3.3M NaCl) onice for two hours. Precipitated phage was spun at 9,100 g at 4° C. for30 minutes and re-suspended in 150 mM NaCl, 10 mM HEPES pH 7.4. Phagewere then re-precipitated as above and suspended in 150 mM NaCl, 10 mMHEPES pH 7.4. Rounds 2 and 3: Amplified phage from Round 1 was incubatedon a tissue culture well without Aβ₁₋₄₀ peptide for one hour in phagebuffer. Supernatant from this well was then incubated on a plate coatedwith amyloid Aβ1-40 as in Round 1. All other procedures were done asdescribed in Round 1.

DNA sequencing of phage clones: At the end of Round 3 of panning,phage-infected K91 cells were diluted and plated as single colonies onLB-Agar plates with 75 μg/ml kanamycin. Individual colonies were grownup at 37 C for 12 hours in LB with 1 μg/ml kanamycin. Bacteria were spundown at 10,000 g for five minutes, after which the supernatant wasprecipitated in PEG/NaOAc (3.6%/450 mM) for 24 hours on ice.Precipitated phage DNA was isolated by spinning at 10,000 g for 15minutes and re-suspended in Tris-EDTA (TE, pH 7.5). Anti-phage primerwas added (5′ gtttgtcgtctttccagacg) (SEQ ID NO:29) and DNA sequencingreactions were run using the Sequenase sequencing kit (Amersham,Piscataway, N.J.).

Staining and blotting with phage: 10¹¹ PFU (plaque forming units)/ml ofvarious phage clones were incubated with the amyloid form of Aβ₁₋₄₀peptide. Aβ amyloid was made by incubating peptide in TBS (pH 7.4) for 7days on Falcon 24-well tissue culture wells. After washing, bound phagewere visualized by incubation with biotinylated anti-M13 antibody andalkaline phosphatase-conjugated streptavidin, followed by incubation forequivalent times in 5-bromo-4-chloro-3-indolyl phosphate (BCIP) andnitroblue tetrazolium (NBT). No staining was seen with any phage oncontrol plates lacking Aβ₁₋₄₀.

Phage were also used to stain monomeric and amyloid forms of Aβ₁₋₄₀peptide that had been immobilized on nitrocellulose. The linear andamyloid forms of Aβ were made as previously described (Tucker et al.,2000) and immobilized nitrocellulose-coated tissue culture plates, alsoas previously described (Martin and Sanes, 1997). Phage clones selectedto bind the amyloid form of Aβ1-40 did not bind non-amyloid Aβ₁₋₄₀. Forimmunoblotting, 10 ng of monomeric Aβ₁₋₄₀ was separated on a 4-12%Bis/Tris NuPAGE gradient gel (Novex; San Diego, Calif.). After transferto nitrocellulose, blots were blocked in phage buffer and incubated with10¹¹ PFU/ml of each clone. After washing in phage buffer (without BSA),blots were incubated with biotinylated anti-M13 antibody followed bystreptavidin-coupled horseradish peroxidase. Blots were developed usingthe ECL chemiluminescence method (Pierce, Madison Wis.). Aβ₁₋₄₀ peptidewas also blotted with a monoclonal antibody that recognizes the Aβpeptide to confirm protein transfer.

Production of recombinant A.beta.-binding peptide: Complementaryoligonucleotides encoding the DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2) peptidesequence were annealed, digested with Kpn I and Xba I, and ligated intopThioHisC plasmid (Invitrogen; Carlsbad, Calif.) at the Kpn I and Xba Isites using the following sequences:

5′ CGGGGTACCTGCAGAATGCGATTGGGGGAAGGGGGGTC (forward)GGTGGCGGTTGTGGCCGGGTGCGTCGGGGAAGACGGAGGCG TGCGGCCCGCCGTATTAGTCTAGAGC(SEQ ID NO: 30) and 5′ GCTCTAGACTAATACGGCGGGCCGCACGCCTCCGTCTT (reverse)CCCCGACGCACCCGGCCACAACCGCCACCGACCCCCCTTCC CCCAATCGCATTCTGCAGGTACCCCG(SEQ ID NO: 31).This added several flanking amino acids from the phage coat sequence ateither end of the 20 amino acid insert, such that the sequence aroundthe insert site (beginning at the Kpn I site in pHisThioC) wasPAEC-insert (DWGKGGRWRLWPGASGKTEA)-CGPPY (SEQ ID NO:32)-stop. XL1-Bluebacteria were transformed with plasmid either containing insert (pThio,Aβ) or lacking insert (pThio). Transformed bacteria were grown andprotein induced with 1 mM IPTG in log phase. Cells were pelleted andharvested by repeated cycles of freezing and thawing with subsequentsonication. Recombinant Thio or Thio-Aβ protein was purified byincubation with ProBond nickel chelating resin (Invitrogen; Carlsbad,Calif.). Protein was bound with pH 7.8 buffer, washed successively withpH 6.0 and pH 5.5 buffers, and eluted with pH 4.0 buffer according tothe manufacturer's instructions. Eluted protein was immediately re-pHedto 7.5 after elution. Recombinant protein comprised ca. 80% of theprotein in fractions used for binding studies.

Binding assays with recombinant Thio and Thio-Aβ protein and syntheticpeptides: Aβ₁₋₄₀ was immobilized on 96-well ELISA plates as describedabove for phage panning. Immobilized Aβ₁₋₄₀ was blocked in phage panningbuffer (Hanks balanced salts with 1 mM CaCl₂ and 1 mM MgCl₂, 10 mMHepes, pH 7.4, and 0.5% BSA) for one hour. Thio or Thio-Aβ protein wasadded at varying concentrations in phage panning buffer for 2 hours atroom temperature. Plates were extensively washed in phage panning bufferwithout BSA. Anti-Thio antibody (Invitrogen; Carlsbad, Calif.) was addedfor 30 minutes in phage panning buffer at a dilution of 1:500. Afterwashing, anti-mouse IgG conjugated to alkaline phosphatase was added at1:500 for 40 minutes. Plates were washed again and incubated withparanitrophenyl phosphate (Sigma; St. Louis, Mo.). Developing substratewas read several times at 405 nm on an ELISA plate reader over thelinear range (OD 0.2-1.0) and normalized to the highest binding signal.To calculate dissociation constants, binding curves were fitted bynon-linear regression analysis assuming a single class of equivalentbinding sites. Binding of primary and secondary antibody to Aβ₁₋₄₀ neverexceeded 10% of the maximal signal for Thio-Aβ in any experiment, andThio protein never bound significantly above primary and secondaryantibody alone at any concentration below 100 μM. Thio did show somebinding in the mM range. By contrast, Thio-Aβ protein was saturating at200 nM.

For binding to synthetic peptides, two peptides were synthesized andpurified containing an N-terminal biotin. One of these was the 20 aminoacid-insert without any flanking sequences, Biotin-DWGKGGRWRLWPGASGKTEA(SEQ ID NO:2). The other sequence, Biotin-AECDWGKGGRWRLWPGASGKTEACGP(SEQ ID NO:4), contained flanking cysteine residues and several otheramino acids from the bacterial coat sequence. These peptides werepurified by HPLC and confirmed by mass spectrometry to be over 90% pure.Peptides were solubilized in phage buffer and incubated at varyingconcentrations with Aβ₁₋₄₀ amyloid for 1 hour. After washing in phagebuffer as above, streptavidin conjugated to alkaline phosphatase wasadded at 1 U/ml for 50 minutes. Plates or slides were washed extensivelyin phage buffer and developed, as above.

Staining of human brain with recombinant Thio-Aβ-protein and syntheticpeptides: Normal and AD brain samples were obtained from the UCSDAlzheimer's Research Center (La Jolla, Calif.). Sections from a non-ADamyloidosis patient were obtained from the Department of Pathology(UCSD). Paraffin-embedded samples of cortex or other tissues weresectioned at 10 μm and mounted on glass slides. After deparifinization,sections were fixed in formic acid, blocked in phage panning buffer, andincubated with 5-500 nM Thio-Aβ or Thio for two hours at roomtemperature. Binding was determined by subsequent staining withanti-Thio antibody and anti-mouse IgG coupled to alkaline phosphatase asabove. After washing, staining was developed using5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium foridentical periods of time. All washes and incubations were done in phagebuffer. The existence of amyloid plaques was confirmed by binding ofanti-Aβ monoclonal antibody (2066) to sections from the same brainsamples. No significant staining was ever observed with secondaryantibody alone. Background staining was allowed to develop to the pointwhere cells in the section were evident. Staining with Thio-Aβ wasconfirmed in brains from multiple AD subjects and was negative inmultiple non-AD controls.

For staining with synthetic peptide, tissue sections from AD and non-ADbrain, as well as from brain, kidney, small bowel, large bowel, andprostate from a non-AD patient with amyloidosis, were cut and preparedas above. Biotinylated peptide (AECDWGKGGRWRLWPGASGKTEACGP) (SEQ IDNO:4) was added in phage buffer for 1 hour at concentrations rangingfrom 0.1-10 μM. Sections were washed with phage buffer, incubated withstreptavidin coupled to alkaline phosphatase, washed and developed asabove. Positive staining of peptide to plaques in AD brain (and negativestaining of non-Aβ amyloid) was confirmed by simultaneous staining ofsections using the same reagents. Congo Red or hematoxylin and eosinstaining was done to confirm the presence of amyloid in amyloidosissections.

Uptake by Neuronal Tissue: The peptides disclosed herein arehydrophobic, have a compact tertiary structure, and bind to the amyloidform of Aβ₁₋₄₀. In addition, the peptides cross the blood-brain barrierefficiently (see FIG. 8).

100 μL of a 1 mg/mL solution of amyloid binding-peptide(biotin-AECDWGKGGRWRLWPGASGKTEACGP (SEQ ID NO:4)) or control peptidewithout cysteines (biotin-DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2)) wasinjected via the tail vein into 8-9 month old wild type CB6 mice.Peptides were injected in sterile Tris-buffered saline, pH 7.4, with 1mM CaCl₂ and 1 mM MgCl₂. 4 animals were injected for each condition.Animals were then sacrificed after one minute or two minutes.Immediately upon sacrifice, blood and organs were harvested. Toquantitate peptide uptake, brain, liver, and kidney were washed and thetissues lysed by homogenization in doubly distilled water. Organs andblood were centrifuged at 13000 g to collect lysate. Organ lysates orserum were then immobilized on ELISA plates that had been coated withnitrocellulose. Some tissue and serum samples were spiked with knownamounts of purified peptide to verify levels of peptide immobilizationin different sample types. For histochemical detection of biotinylatedpeptides in brain, a portion of the brain from each experiment wasdissected and fixed for one day in 4% paraformaldehyde. These tissueswere then dehydrated in PBS with varying levels of sucrose, frozen, andsectioned as described in Hoyte et al. (Brain Research: Molecular BrainResearch 109:146-160 (2002)). 8 mm brain sections were stained forbiotinylated peptide by binding of alkaline phosphatase-conjugatedstreptavidin. Levels of streptavidin binding were determined bydevelopment of sections in nitroblue tetrazolium and5-bromo-4-chloro-3-indolyl phosphate. The levels of biotinylated peptideimmobilized on ELISA plates was quantitated by binding of alkalinephosphatase-conjugated streptavidin. Levels of streptavidin binding weredetermined by development with para-nitrophenylphosphate, as in Kang etal. (Neurobiology of Disease 14:146-156 (2003)).

Results of the rate of uptake, per minute, as a percentage relative tothe amount of peptide in serum, is provided in Table 2 and FIG. 8. Theamyloid binding peptide crossed the blood brain barrier into the brainparenchyma at a rate that was equivalent to that measured in kidney andat a rate that exceeded uptake into the liver.

In contrast, non-disulfide containing control peptide did not cross theblood-brain barrier. Access of the amyloid binding peptide was confirmedby immunostaining of sagittal sections of the brain. Here, both whiteand grey matter were positively stained, while the control peptidelacking cysteines only stained blood vessels (FIG. 8). These resultsindicate that the amyloid binding peptides disclosed herein can enterthe brain.

The data demonstrate a practical way in which the peptides of theinvention can be utilized as diagnostic or therapeutic agent in patientsthat have, or are at risk to develop, Alzheimer's disease. The fact thata rather large side chain, biotin, can be coupled to the peptide andthat this does not create a barrier to brain uptake demonstrates theutility of this peptide to deliver conjugated agents. Such agents couldbe contrast agents to allow imaging of amyloid plaques, such asgadolinium, which would allow imaging by MRI, or therapeutic agents.

As indicated by the data presented in Table 2, one could achievetherapeutic and/or diagnostic concentrations of a peptide of theinvention in the brain within 5 minutes by injecting 5-28 μg of peptidein the mouse or 3.7-21 mg of peptide in humans intravenously. Thiscalculation assumes a cerebrospinal fluid volume of 200 μl in the mouseand 150 ml in the human. This is very practical amount of material thatcould be synthesized on a commercial scale. Thus, the robust nature ofthe uptake of this peptide into the brain should allow concentrations tobe reached that would identify amyloid plaques. In addition, the robustnature of its uptake into the brain should allow for considerableflexibility in the types of reagents with which it could be modified.

FIG. 1 shows staining of amyloid Aβ₁₋₄₀ by phage peptides. Both phagepeptide sequences selected for Aβ₁₋₄₀ amyloid binding stained amyloiddeposits in vitro. Final clones 2 and 4 are the DWGKGGRWRLWPGASGKTEA(SEQ ID NO:2) sequence. This sequence identified both small and large(0.5-50 μm) accumulations of Aβ₁₋₄₀ amyloid. Final clone 6 is thePGRSPFTGKKLFNQEFSQDQ (SEQ ID NO:3) sequence. This sequence stainedAβ₁₋₄₀ amyloid aggregates more poorly, but still stained well abovebackground levels. None of the ten starting clones randomly picked(Starting clone 6 is shown) stained when used at the same concentration.

FIG. 2 shows immunoblotting of monomeric Aβ₁₋₄₀ by phage peptides.Monomeric Aβ₁₋₄₀ was separated on a 4-12% Bis/Tris gradient gel andblotted with either anti-Aβ antibody or with phage clones. No startingor final phage peptide clones recognized monomeric Aβ₁₋₄₀ peptide.Blotting with a monoclonal antibody that recognizes Aβ₁₋₄₀ is shown as acontrol for protein transfer.

FIG. 3 shows recombinant Aβ-binding peptide binds with high affinity toAβ₁₋₄₀ amyloid in vitro. A recombinant cysteine-linked form of theDWGKGGRWRLWPGASGKTEA (SEQ ID NO:2) sequence was produced as a fusionprotein with thioredoxin in E. coli (Thio-Aβ). RecombinantThio-A.beta.was purified and binding to Aβ₁₋₄₀ amyloid was measured.Recombinant Thio-Aβ bound Aβ₁₋₄₀ amyloid with a Kd of 60 nM. Binding wassaturating by 200 nM. Recombinant purified thioredoxin (Thio) showed nobinding at any of the concentrations used. Errors are SEM for n=6.

FIG. 4 shows specific binding of Thio-Aβ to amyloid plaques inAlzheimer's disease brain. Recombinant Aβ-binding peptide conjugated tothioredoxin (Thio-Aβ) was used to stain brain samples from normalsubjects and those with Alzheimer's disease (AD). Thio-Aβ did not stainany structures in normal brain tissue, but heavily stained amyloidplaques from AD brains. Background staining was allowed to increase toallow visualization of cells within the section, but this staining wasnot caused by Thio-Aβ. Thioredoxin (Thio) did not stain amyloid plaquesin AD brains when added at the same concentrations. Bar is 100 μm forpanels on the left and 25 μm for panels on the right.

FIG. 5 shows binding of synthetic peptides to Aβ₁₋₄₀ amyloid in vitro.Two biotin-labeled peptides, Biotin-DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2)and Biotin-AECDWGKGGRWRLWPGASGKTEACGP (SEQ ID NO:4), were tested forbinding to Aβ₁₋₄₀ amyloid. The peptide containing flanking cysteinesbound with a Kd of 320 nM, while the peptide lacking these cysteines didnot bind with significant affinity below 5 μM. Errors are SEM for n=6.

FIG. 6 shows specific staining amyloid plaques in Alzheimer's diseasebrain with a synthetic peptide. The Biotin-AECDWGKGGRWRLWPGASGKTEACGPpeptide was used to stain brain sections from normal and AD brain. Thispeptide specifically stained amyloid plaques in AD brain. The peptidedid not stain non-Aβ amyloid in tissues from a patient with amyloidosis(kidney is shown). Bar is 100 μm for panels on the left and 25 μm forpanels on the right.

FIG. 7 shows a model of how Aβ1-40 binding-peptides can be used. Thecysteine-linked peptide sequences CDWGKGGRWRLWPGASGKTEAC (SEQ ID NO:5)and CPGRSPFTGKKLFNQEFSQDQC(SEQ ID NO:6) can be used to bind to amyloidplaques in Alzheimer's disease brain. These peptides could be used ascarriers to deliver molecules to amyloid plaques that 1) lessen theirneurotoxicity, 2) stimulate their destruction, or 3) inhibit theirformation. In addition, such peptides could be conjugated to moleculesused to 4) visualize amyloid plaques, or 5) induce an anti-idiotypeantibody.

FIG. 8 depicts entry of amyloid binding peptide into the brain afterintravenous injection. Amyloid binding peptide(biotin-AECDWGKGGRWRLWPGASGKTEACGP (SEQ ID NO:4)) was compared tobinding of a control peptide lacking cysteines(biotin-DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2)) 2 minutes after intravenousinjection via the tail vein in wild type mice. Control peptide waspresent in some blood vessels, but did not enter the brain parenchyma.Amyloid binding peptide, by contrast, entered the brain parenchyma inlarge amounts. Sagittal section of cortex is shown. The bar indicates100 mm in A, B, 50 mm in C, D.

Table 1: Identification of peptides that adhere to Aβ₁₋₄₀ amyloid. Arandom 20-amino acid cysteine-cross-linked phage peptide library with5×10⁷ possible sequences was screened for adhesion to Aβ₁₋₄₀ amyloid.Sequences of 10 randomly picked phage clones in the starting library areshown, as are sequences of 10 randomly picked phage clones isolatedafter three rounds of panning against Aβ₁₋₄₀ amyloid. At least twopeptides adhered to Aβ₁₋₄₀ amyloid. These sequences shared a density ofsimilarly spaced bulky hydrophobic amino acids (underlined) that werenot present in clones picked from the starting library. Two positivelycharged amino acids (dark) were present between the first twohydrophobic residues in both peptides.

Random Starting Clone Sequences:

1. LGSGRIGDGWSDGGLARRLK (SEQ ID NO:7) 2. DGGGGAGRWTTKDRSAAKTE (SEQ IDNO:8) 3. VDDGAQGKRWGGMGLGKGRR (SEQ ID NO:9) 4. SGSGVGLRMASQRHEGRKVY (SEQID NO:10) 5. QLPQNGGPAWFTRKAGQGGR (SEQ ID NO:11) 6. LGYAGGGQGMVEGSFWPTSW(SEQ ID NO:12) 7. GLRGMEGRGYPKDRRDRNLE (SEQ ID NO:13) 8.LIGGNKAGRGAWGVVASSGR (SEQ ID NO:14) 9. ELESRGGLGYAWRGSASTMD (SEQ IDNO:15) 10. KGETGNGGRAKAGTVDLIRR (SEQ ID NO:16)Random Final Clone Sequences:

1. DWGKGGRWRLWPGASGKTEA (SEQ ID NO: 2) 2. DWGKGGRWRLWPGASGKTEA (SEQ IDNO: 2) 3. DWGKGGRWRLWPGASGKTEA (SEQ ID NO: 2) 4. DWGKGGRWRLWPGASGKTEA(SEQ ID NO: 2) 5. DWGKGGRWRLWPGASGKTEA (SEQ ID NO: 2) 6.PGRSPFTGKKLFNQEFSQDQ (SEQ ID NO: 3) 7. DWGKGGRWRLWPGASGKTEA (SEQ ID NO:2) 8. PGRSPFTGKKLFNQEFSQDQ (SEQ ID NO: 3) 9. DWGKGGRWRLWPGASGKTEA (SEQID NO: 2) 10. DWGKGGRWRLWPGASGKTEA (SEQ ID NO: 2)

Table 2: Rate of uptake of amyloid binding peptide in brain, kidney, andliver. The rate of uptake of amyloid binding peptide was quantitated asa percentage of the concentration delivered intravenously into serum andcompared to that for a non-cysteine containing control peptide. Only theamyloid binding peptide containing cysteines was delivered into thebrain at a significant rate, and was equivalent to the rate of uptakeinto the kidney or the liver. Errors are SD for n=4.

Organ Peptide (SEQ ID NO:) % uptake/min Brain biotin-AECDWGKGGRWRLWPGA 0.18 ± 0.02% SGKTEACGP (4) Liver biotin-AECDWGKGGRWRLWPGA  0.06 ± 0.02%SGKTEACGP (4) Kidney biotin-AECDWGKGGRWRLWPGA  0.16 ± 0.02% SGKTEACGP(4) Brain biotin-DWGKGGRWRLWPGASG  0.00005 ± 0.00005% KTEA (2)It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

1. An isolated polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:5.
 2. An isolated polypeptide consisting of the amino acid sequence as set forth in SEQ ID NO:5.
 3. The isolated polypeptide of claim 1 or 2, wherein the cysteine residues are intramolecularly cross-linked via a disulfide bond.
 4. The isolated polypeptide of claim 1, further comprising from 1 to 15 additional amino acids at the N- or C-terminus of the polypeptide comprising SEQ ID NO:5, wherein the polypeptide interacts with the amyloid form of the Aβ peptide comprising the Aβ1-40 peptide.
 5. The isolated polypeptide of claim 1, further comprising from 1 to 10 additional amino acids at the N- or C-terminus of the polypeptide comprising SEQ ID NO:5, wherein the polypeptide interacts with the amyloid form of the Aβ peptide comprising the Aβ1-40 peptide.
 6. The isolated polypeptide of claim 1, further comprising from 1 to 5 additional amino acids at the N- or C-terminus of the polypeptide comprising SEQ ID NO:5, wherein the polypeptide interacts with the amyloid form of the Aβ peptide comprising the Aβ1-40 peptide.
 7. The isolated polypeptide of claim 1, further comprising from 1 to 3 additional amino acids at the N- or C-terminus of the polypeptide comprising SEQ ID NO:5, wherein the polypeptide interacts with the amyloid form of the Aβ peptide comprising the Aβ1-40 peptide.
 8. The isolated polypeptide of claim 7, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4 or consists of the amino acid sequence of SEQ ID NO:4.
 9. The polypeptide of claims 1, 2 or 8, wherein the polypeptide binds to the amyloid form of the Aβ peptide comprising Aβ 1-40 peptide.
 10. The polypeptide of claim 1, further comprising a detectable label conjugated to the polypeptide, wherein the detectable label is selected from the group consisting of a fluorescent moiety, a chromogenic moiety, a luciferase, biotin and a radiolabel.
 11. A composition for detecting Aβ peptide comprising Aβ1-40 peptide in a mammal comprising an effective amount of the polypeptide of claim
 10. 12. A hybrid molecule comprising: a) the peptide set forth in claim 1, 2 or 8, that specifically interacts with the amyloid form of the Aβ peptide comprising the Aβ1-40 peptide; and b) a diagnostic or therapeutic reagent detectable label conjugated to the polypeptide, wherein the detectable label is selected from the group consisting of a fluorescent moiety, a chromogenic moiety, a luciferase, biotin and a radiolabel.
 13. A method of detecting Aβ peptide comprising Aβ1-40 peptide in a mammal comprising administering to the mammal the composition of claim
 11. 